Restriction endonucleases are a class of enzymes that occur naturally in prokaryotic and eukaryotic organisms. When they are purified away from other contaminating cellular components, restriction endonucleases can be used in the laboratory to cleave DNA molecules into precise fragments. This property enables DNA molecules to be uniquely identified and to be fractionated into their constituent genes. Restriction endonucleases have proved to be indispensable tools in modern genetic research. They are the biochemical "scissors" by means of which genetic engineering and analysis are performed.
Restriction endonucleases act by recognizing and binding to particular sequences of nucleotides (the "recognition sequence") along the DNA molecule. Once bound, they cleave the molecule within, or to one side of, this sequence in both strands. Different restriction endonucleases have affinity for different recognition sequences. About 100 kinds of different endonucleases have so far been isolated from many microorganisms, each being identified by the specific base sequence it recognizes and by the cleavage pattern it exhibits. In addition, a number of restriction endonucleases, called restriction endonuclease isoschizomers, have been isolated from different microorganisms which in fact recognize the same recognition sequence as those restriction endonucleases that have previously been identified. These isoschizomers, however, may or may not cleave the same phosphodiester bond as the previously identified endonuclease.
In nature, restriction endonucleases play a protective role in the welfare of the microbial cell. They enable the microorganism to resist infection by foreign DNA molecules like viruses and plasmids that would otherwise destroy or parasitize them. They achieve this resistance by scanning the lengths of the infecting DNA molecule and cleaving them each time that the recognition sequence occurs. The DNA cleavage that takes place disables many of the infecting genes and renders the DNA susceptible to further degradation by nonspecific exonucleases.
A second component of microbial protective systems are the modification methylases. Modification methylases are complementary to their corresponding restriction endonucleases in that they recognize and bind to the same recognition sequence. Modification methylases, in contrast to restriction endonucleases, chemically modify certain nucleotides within the recognition sequence by addition of a methyl group. Following this methylation, the recognition sequence is no longer bound or cleaved by the restriction endonuclease. The microbial cell modifies its DNA by virtue of its modification methylases and therefore is completely insensitive to the presence of its endogenous restriction endonucleases. Thus, endogenous restriction endonuclease and modification methylase provide the means by which a microorganism is able to identify and protect its own DNA, while destroying unmodified foreign DNA.
The combined activities of the restriction endonuclease and the modification methylase are referred to as the restriction-modification system. Three types of restriction-modification systems have been identified that differ according to their subunit structure, substrate requirements and DNA cleavage. Specifically, Type-I and Type-III restriction systems carry both modification and ATP-requiring restriction (cleavage) activity in the same protein. Type-II restriction-modification systems, on the other hand, consist of a separate restriction endonuclease and modification methylase, i.e., the two activities are associated with independent proteins.
Type II restriction endonucleases are endodeoxyribonucleases which are commonly used in modern genetic research. These enzymes recognize and bind to particular DNA sequences and once bound, cleave within or near this recognition sequence. Phosphodiester bonds are thereby hydrolyzed in the double stranded DNA target sequence, i.e., one in each polynucleotide strand. Type-II restriction endonucleases can generate staggered breaks within or near the DNA recognition sequence to produce fragments of DNA with 5' protruding termini, or DNA fragments with 3' protruding termini. Other Type-II restriction endonucleases which cleave at the axis of symmetry, produce blunt ended DNA fragments. Therefore, Type-II restriction endonucleases can differ according to their recognition sequence and/or the location of cleavage within that recognition sequence.
Type-II restriction endonucleases are frequently used by the genetic engineers to manipulate DNA in order to create novel recombinant molecules. Specific Type-II restriction endonucleases are known for numerous DNA sequences, but there is still a need to provide new Type-II restriction endonucleases. These new enzymes will add to the list of indispensable tools needed for modern genetic research.